The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.     

Conjugal blocks in Tetrahymena pattern mutants and their cytoplasmic rescue. I. Broadened cortical domains (bcd).

The cortical pattern mutant broadened cortical domains (bcd) in Tetrahymena thermophila is unable to complete the nuclear events associated with conjugation. bcd x bcd pairs become arrested at the "nuclear exchange" configuration. Genetic analysis reveals that the bcd conjugal block is 100% penetrant, under macronuclear control, and rescueable (a) by outcrossing to a wild-type partner, (b) by administration of a hyperosmotic shock 5 hr after cells are mixed for mating, or (c) by cytoplasmic transfusion from a wild-type donor. Cytological analysis reveals that the conjugal block is primarily the result of failure in pronuclear fusion (karyogamy). bcd pairs also exhibit reduced nuclear exchange efficiency and a failure of macronuclear anlagen formation. The hypothesis is proposed that the bcd+ gene codes for a microtubule-based organelle "motor" similar to kinesin.     

Phylogenetic analysis of the pathogenic anaerobe Clostridium perfringens using the 16S rRNA nucleotide sequence.

Clostridium perfringens, the first pathogenic clostridium examined, was placed in the nonmycoplasma subgroup of the low-dG+dC-content gram-positive cluster on the basis of the results of a phylogenetic analysis in which we used 16S rRNA comparisons. The closest relative that has been identified to date is Clostridium pasteurianum.     

1.7 A X-ray structure of the periplasmic ribose receptor from Escherichia coli.

The X-ray structure of the periplasmic ribose receptor (binding protein) of Escherichia coli (RBP) was solved at 3 A resolution by the method of multiple isomorphous replacement. Alternating cycles of refitting and refinement have resulted in a model structure with an R-factor of 18.7% for 27,526 reflections from 7.5 to 1.7 A resolution (96% of the data). The model contains 2228 non-hydrogen atoms, including all 271 residues of the amino acid sequence, 220 solvent atoms and beta-D-ribose. The protein consists of two highly similar structural domains, each of which is composed of a core of parallel beta-sheet flanked on both sides by alpha-helices. The two domains are related to each other by an almost perfect 2-fold axis of rotation, with the C termini of the beta-strands of each sheet pointing toward the center of the molecule. Three short stretches of amino acid chain (from symmetrically related portions of the protein) link these two domains, and presumably act as a hinge to allow relative movement of the domains in functionally important conformational changes. Two water molecules are also an intrinsic part of the hinge, allowing crucial flexibility in the structure. The ligand beta-D-ribose (in the pyranose form) is bound between the domains, held by interactions with side-chains of the interior loops. The binding site is precisely tailored, with a combination of hydrogen bonding, hydrophobic and steric effects giving rise to tight binding (0.1 microM for ribose) and high specificity. Four out of seven binding-site residues are charged (2 each of aspartate and arginine) and contribute two hydrogen bonds each. The remaining hydrogen bonds are contributed by asparagine and glutamine residues. Three phenylalanine residues supply the hydrophobic component, packing against both faces of the sugar molecule. The arrangement of these hydrogen bonding and hydrophobic residues results in an enclosed binding site with the exact shape of the allowed sugar molecules; in the process of binding, the ligand loses all of its surface-accessible area. The sites of two mutations that affect the rate of folding of the ribose receptor are shown to be located near small cavities in the wild-type protein. The cavities thus allow the incorporation of the larger residues in the mutant proteins. Since these alterations would seriously affect the ability of the protein to build the first portion of the hydrophobic core in the first domain, it is proposed that this process is the rate-limiting step in folding of the ribose receptor.     

Inhibition of HIV-1 IIIb replication in AA-2 and MT-2 cells in culture by two ligands of poly (ADP-ribose) polymerase: 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone

The effects of two adenosine diphosphoribose transferase (ADPRT) enzyme inhibitory ligands, 6-amino-1,2-benzopyrone and its 5-iodo-derivative, were determined in AA-2 and MT-2 cell cultures on the replication of HIV-1 IIIb, assayed by an immunochemical test for the HIV protein p24, and syncytium formation, characteristic of HIV-infected cells. Intracellular concentrations of both drugs were sufficient to inhibit poly(ADP-ribose) polymerase activity within the intact cell. Both drugs inhibited HIV replication parallel to their inhibitory potency on ADPRT, but distinct differences were ascertained between the two cell lines. In AA-2 cells both p24 and syncytium formation were depressed simultaneously, whereas in MT-2 cells only syncytium formation was inhibited by the drugs, and the p24 production, which remained unchanged during viral growth, was unaffected. Both drugs only moderately depressed the growth rate of the AA-2 and MT-2 cells and there was no detectable cellular toxicity. Results suggest the feasibility of the development of a new line of ADPRT ligand anti-HIV drugs that fundamentally differ in their mode of action from currently used chemotherapeutics.     

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.     

Spatial constraints on polyadenylation signal function

Efficient cleavage and polyadenylation of eukaryotic messenger RNAs require at least two signal elements: an AAUAAA or closely related sequence located 7-30 base pairs (bp) upstream of the site of processing, and a G/U- or U-rich sequence located 3' to the cleavage site. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT-rich region. We have shown that the first AATAAA and the GT-rich region are essential for efficient processing, both in vivo and in vitro, whereas the second AATAAA does not appear to play any role in the formation of tk mRNA 3' ends. The failure of a signal containing only the second AATAAA and the GT-rich element to signal cleavage and polyadenylation suggested that these two elements might be too close together to constitute a functional polyadenylation signal. The experiments described in this report were directed at determining the effects on mRNA 3' end formation of alterations in spacing between signal elements. Wild-type tk contains 19 bp between these two elements. Constructs were made in which an AATAAA and the GT-rich region were separated by various distances ranging from 7 to 43 bp. The quantity and location of 3' ends of the tk mRNA produced by these constructs in Cos-1 cells were measured by S1 nuclease protection analysis. Signal efficiency was gradually reduced as the separation between the two signal elements was increased; with a separation of 43 bp, the signal functioned at approximately one-eighth the efficiency of the parental construction. Bringing the two signals closer together resulted in decreased signal efficiency; with a separation of 7 or 9 bp, no tk mRNA polyadenylated within the normal region was produced. Altering the sequences between these two elements without changing the distance had small effects on processing efficiency.     

The S3 state of photosystem II: differences between the structure of the manganese complex in the S2 and S3 states determined by X-ray absorption spectroscopy

O2-evolving photosystem II (PSII) membranes from spinach have been cryogenically stabilized in the S3 state of the oxygen-evolving complex. The cryogenic trapping of the S3 state was achieved using a double-turnover illumination of dark-adapted PSII preparations maintained at 240 K. A double turnover of PSII was accomplished using the high-potential acceptor, Q400, which is the high-spin iron of the iron-quinone acceptor complex. EPR spectroscopy was the principal tool establishing the S-state composition and defining the electron-transfer events associated with a double turnover of PSII. The inflection point energy of the Mn X-ray absorption K-edge of PSII preparations poised in the S3 state is the same as for those poised in the S2 state. This is surprising in light of the loss of the multiline EPR signal upon advancing to the S3 state. This indicates that the oxidative equivalent stored within the oxygen-evolving complex (OEC) during this transition resides on another intermediate donor which must be very close to the manganese complex. An analysis of the Mn extended X-ray absorption fine structure (EXAFS) of PSII preparations poised in the S2 and S3 states indicates that a small structural rearrangement occurs during this photoinduced transition. A detailed comparison of the Mn EXAFS of these two S states with the EXAFS of four multinuclear mu-oxo-bridged manganese compounds indicates that the photosynthetic manganese site most probably consists of a pair of binuclear di-mu-oxo-bridged manganese structures. However, we cannot rule out, on the basis of the EXAFS analysis alone, a complex containing a mononuclear center and a linear trinuclear complex. The subtle differences observed between the S states are best explained by an increase in the spread of Mn-Mn distances occurring during the S2----S3 state transition. This increased disorder in the manganese distances suggests the presence of two inequivalent di-mu-oxo-bridged binuclear structures in the S3 state.